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Live‐imaging of revertant and therapeutically restored dystrophin in the Dmd EGFP‐mdx mouse model for Duchenne muscular dystrophy
Author(s) -
Petkova M. V.,
Stantzou A.,
Morin A.,
Petrova O.,
MoralesGonzalez S.,
Seifert F.,
BellecDyevre J.,
Manoliu T.,
Goyenvalle A.,
Garcia L.,
Richard I.,
LaplaceBuilhé C.,
Schuelke M.,
Amthor H.
Publication year - 2020
Publication title -
neuropathology and applied neurobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.538
H-Index - 95
eISSN - 1365-2990
pISSN - 0305-1846
DOI - 10.1111/nan.12639
Subject(s) - dystrophin , duchenne muscular dystrophy , mdx mouse , ex vivo , utrophin , green fluorescent protein , microbiology and biotechnology , biology , exon skipping , muscular dystrophy , exon , in vivo , genetics , gene , alternative splicing
Background Dmd mdx , harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies. Methods We generated a Dmd EGFP‐mdx reporter allele carrying in cis the mdx‐ 23 mutation and a C‐terminal EGFP‐tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin‐EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo‐DNA to skip Dmd exon 23, or after gene editing using AAV‐encoded CRISPR/Cas9 for Dmd exon 23 excision. Results Intravital microscopy in anaesthetized mice allowed live‐imaging of sarcolemmal dystrophin‐EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin‐EGFP‐fluorescence persisted ex vivo , allowing live‐imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo . Expression of the shorter dystrophin‐EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx‐ 23 mutation. Conclusion Intravital imaging of Dmd EGFP‐mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo .